Download Repeated evolution of reproductive isolation in a marine snail

Phil. Trans. R. Soc. B (2010) 365, 1735–1747
doi:10.1098/rstb.2009.0256
Repeated evolution of reproductive isolation
in a marine snail: unveiling mechanisms
of speciation
Kerstin Johannesson1,*, Marina Panova1, Petri Kemppainen1,
Carl André1, Emilio Rolán-Alvarez2 and Roger K. Butlin3
1
Department of Marine Ecology—Tjärnö, University of Gothenburg, 452 96 Strömstad, Sweden
Departemento de Bioquı́mica, Genética e Immunologı́a, Facultad de Biologı́a, Universidad de Vigo,
Campus As Lagoas-Marcosende, 36310 Vigo, Spain
3
Department of Animal and Plant Sciences, University of Sheffield, Western Bank,
Sheffield S10 2TN, UK
2
Distinct ecotypes of the snail Littorina saxatilis, each linked to a specific shore microhabitat, form a
mosaic-like pattern with narrow hybrid zones in between, over which gene flow is 10 –30% of
within-ecotype gene flow. Multi-locus comparisons cluster populations by geographic affinity independent of ecotype, while loci under selection group populations by ecotype. The repeated
occurrence of partially reproductively isolated ecotypes and the conflicting patterns in neutral
and selected genes can either be explained by separation in allopatry followed by secondary overlap
and extensive introgression that homogenizes neutral differences evolved under allopatry, or by
repeated evolution in parapatry, or in sympatry, with the same ecotypes appearing in each local
site. Data from Spain, the UK and Sweden give stronger support for a non-allopatric model of ecotype formation than for an allopatric model. Several different non-allopatric mechanisms can,
however, explain the repeated evolution of the ecotypes: (i) parallel evolution by new mutations
in different populations; (ii) evolution from standing genetic variation; and (iii) evolution in concert
with rapid spread of new positive mutations among populations inhabiting similar environments.
These models make different predictions that can be tested using comprehensive phylogenetic
information combined with candidate loci sequencing.
Keywords: Littorina saxatilis; ecotype evolution; parallel evolution; selective sweep;
evolution in concert; incipient speciation
1. INTRODUCTION
Reproductive barriers that evolve repeatedly in independent populations offer a replicated experiment
for testing of speciation mechanisms. Stochastic
forces alone cannot explain the repeated patterns of
evolution, and the primary conclusion is that natural
selection is involved (Schluter & Nagel 1995;
Johannesson 2001; Coyne & Orr 2004; Schluter
et al. 2004). Furthermore, reproductive barriers
between host races or ecotypes function in the presence of gene flow, and studies of these types of
barriers are hence more informative to understand
initial steps of speciation than studies of barriers
between already separated species, as the latter barriers
may have accumulated after the completion of
speciation (Coyne & Orr 2004; Via & West 2008;
Schluter 2009). But, of course, one must be aware
that not all partial reproductive barriers evolve to
completion (Nosil et al. 2009).
The evolution of partially reproductively isolated
ecotypes in the seashore periwinkle Littorina saxatilis
has been suggested as an example of parallel evolution
* Author for correspondence ([email protected]).
One contribution of 11 to a Theme Issue ‘Genomics of speciation’.
of reproductive isolation/incipient speciation (RolánAlvarez et al. 2004; Panova et al. 2006; Quesada
et al. 2007; Schluter 2009) showing circumstantial evidence for strong ecological separation being the main
factor behind the evolution of reproductive barriers.
Nevertheless, it has been suggested that these barriers
may, at least in part, be due to genetic differences
accumulated during an earlier period of allopatric separation (Grahame et al. 2006; Butlin et al. 2008).
Indeed, other earlier presumed cases of sympatric speciation have recently been shown to involve
components of allopatric origin, such as the Mexican
inversion in Rhagoletis that contributed to the production of a host-shift (Feder et al. 2003).
Ecotype formation in L. saxatilis provides an excellent model system for studies of both ecological
mechanisms of divergence and origin of genetic variation for ecological adaptation ( Johannesson 2009).
Firstly, microhabitat-specific ecotypes have evolved in
a variety of different rocky shore areas over the complete range of species, providing a test of general
mechanisms under a variety of different circumstances.
Secondly, ecotype formation, including the evolution
of reproductive isolation, can be studied at different
scales. At a local scale, ecotypes are distributed in
such a way that pairs of contrasting ecotypes are
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This journal is q 2010 The Royal Society
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K. Johannesson et al.
Repeated evolution of isolation
present on islands in an archipelago, or on different
parts of the same shore. At a regional scale, similar
ecotypes occur at geographical distances of at least
200 – 400 km. Finally, at distances of 1000 – 3000 km,
ecotypes living under similar selective regimes share
basic phenotypic characteristics such as size, shell
thickness and behaviour (table 1). This gives ample
opportunities to study phenotypic similarities and
divergences at different phylogeographic levels and at
different levels of contemporary gene flow. Thus far,
comprehensive studies of this system have been
undertaken in Spain, the UK and Sweden. Here, we
compare results obtained in the three systems, and
synthesize the current knowledge on the mechanisms
of ecotype formation, in particular the evolution of
reproductive isolation. We suggest four mechanisms
that alone or in various combinations may explain
the repeated evolution of ecotypes in L. saxatilis,
and we outline the genomic signatures that will be
predicted from each of these mechanisms.
2. ON TERMINOLOGY
Recently, Arendt & Reznick (2007) made the case that
the standard use of the two terms ‘parallel evolution’
and ‘convergent evolution’ for independent evolution
of similar traits in closely and distantly related taxa,
respectively, is misleading, as the same gene can sometimes be found to cause the same phenotypic effect in
distantly related species, while at the same time, different genes may give the same effect in populations of
the same species. Furthermore, Butlin et al. (2008)
emphasize the importance of distinguishing between
parallel ecological divergence of ecotypes and independent parallel origin of alleles. The latter is just one
possible mechanism explaining parallel evolution of
ecotypes, host races, etc., but this distinction is
mostly not made in the literature (Schluter & Nagel
1995; Johannesson 2001; Schluter et al. 2004;
Colosimo et al. 2005; Arendt & Reznick 2007;
Schluter 2009). The key feature of parallel evolution
is the repeated evolution of similar phenotypes in independent situations. Independence is critical to be able
to conclude that parallel evolution is a result of selection alone. Indeed, there are alternative processes
that can lead to repeated evolution of phenotypes in
which the genetic variation that selection is acting on
has a common origin, rather than several independent
origins. If this is so, evolution may follow a path that is
partly dictated by the available genetic variation, and
the repeated evolution of similar phenotypes is partly
related to connections between populations. To stress
the importance of independence, here we use the
term ‘repeated evolution of phenotypes’ when the genetic backgrounds of traits are unknown or not
independent, and we use ‘parallel evolution’ when
similar phenotypic traits in different populations have
evolved de novo from independent new mutations
with similar phenotypic effects, or from repeated substitutions of the same nucleotide (Wood et al. 2005).
We make the distinction between allopatry and nonallopatry (parapatry and sympatry) and consider these
terms equivalent to ‘in the absence of gene flow’ or ‘in
the presence of gene flow’, respectively. Following
Phil. Trans. R. Soc. B (2010)
Bolnick & Fitzpatrick (2007) (see also discussions in
Mallet et al. 2009 and Fitzpatrick et al. 2009), we consider the spatial context of speciation to be important,
because it reflects the level of connectivity/independence of populations although we do not expect to
find complete sympatry nor complete allopatry in
nature (Butlin et al. 2008; Fitzpatrick et al. 2008). As
underlined by Rieseberg & Burke (2001), levels of
migration that are much smaller than what is needed
to prevent divergence of populations by drift (i.e.
Nm 1) will be crucial in the process of transferring
positively selected alleles among populations of a
species (or even among species). Unfortunately,
migration rates at these low levels are extremely hard
to measure (Strasburg & Rieseberg 2008).
3. THE BIOLOGY OF LITTORINA SAXATILIS
The rough periwinkle, L. saxatilis, is widely distributed
with dense continuous populations (100 – 1000 individuals per square metre) along wave-impacted rocky
shores, and in lagoons and salt-marshes of the northern Atlantic, from Portugal to Novaya Zemlya and
Svalbard, and from North Carolina to Greenland
(Reid 1996). A few, probably introduced, populations
are present elsewhere (Italy, South Africa, California).
The species forms distinct ecotypes in different microhabitats and gene flow between adjacent ecotypes is
strongly (although not completely) impeded with the
formation of micro-scale hybrid zones (Johannesson
et al. 1993, 1995a; Rolán-Alvarez et al. 1999; Grahame
et al. 2006; Panova et al. 2006). A most important trait
is the lack of migratory (pelagic) larvae resulting in
restricted dispersal among populations. Snails may
either crawl along the shore in the range of 2– 10 m
per generation (Janson 1983; Erlandsson et al. 1998)
or occasionally drift attached to rafting pieces of
macroalgae or frozen up in ice floes or similar.
Migration rates through rafting are hard to assess.
However, after a toxic algal bloom that wiped out all
snails on small islands (at distances of 0.1 – 1 km
from larger islands), the rate of island re-colonization
was 1.5 per cent per generation ( Johannesson &
Johannesson 1995). As a successful recolonization
requires only one adult female (see below), migration
between islands in the archipelago can be estimated
to be in the region of 0.03 migrants (assuming male
and female rafting to be equally frequent) per generation, that is, Nm 1. Obviously, migration over
long geographical distances (greater than 100 –
1000 km) is likely to be much less frequent. Notably,
founding a new population will require no more than
a single mated female as she has the capacity to release
a few hundred offspring that will remain in the area
throughout life. (Although not all offspring survive
until the adult stage, observations after the algal
bloom indicated that populations of re-colonized
islands expanded exponentially and returned to
normal densities in a few generations (Johannesson &
Johannesson 1995).) Hence, one female being successfully rafted to a new site is probably enough to
establish a new population in that site, and this may
explain this species being established in extremely
remote places like the small and isolated island of
Repeated evolution of isolation K. Johannesson et al.
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Table 1. Summary of data gathered from ecotype pairs of Littorina saxatilis in Spain, the UK and Sweden.
Spain
UK
Sweden
ecotype
metapopulation
structure
age of
establishment
morphological
phenotype
RB
SU
coastal and large islands
M
coastal
,40 000 yr BP
,10 000 yr BP
,10 000 yr BP
large,
thickshelled,
narrow
aperture,
ridged
small, thinshelled,
large
aperture,
smooth
large,
thickshelled,
narrow
aperture,
smooth
large,
thickshelled,
narrow
aperture,
smooth
small, thinshelled,
large
aperture,
smooth
behavioural
phenotype
warya
bolda
wary
bold
habitat
upper
intertidal
lower
intertidal
mean
intertidal
boulders
upper
intertidal
vertical
cliffs
intertidal
cliffs
intertidal
boulders
inferred main
selection
hybrid zone
extension
hybrid fitness
crabs
waves
crabs
waves
crabs
waves
H
small, thinshelled,
large
aperture,
smooth
references
S
E
small islands
sympatricb
sympatricb (low density)
parapatric
hybrids as fit as parental
forms in hybrid zone
inconclusive, possibly
postzygotic barrier
bounded hybrid
superiority
evidence of
reproductive
barrier
assortative mating in
microsympatric sites in
the field
assortative mating in
laboratory experiments
assortative mating in
laboratory experiments
neutral gene flow
over hybrid zone
compared with
within-ecotypec
8.8% (allozymes)
19.4% (AFLP)
7.5% (microsatellites)
29.2% (AFLP)
12.7% (microsatellites)
Quesada et al.
(2007)
Johannesson &
Johannesson
(1996);
Hollander et al.
(2006); CondePadin et al.
(2009)
Johannesson &
Johannesson
(1996)
Janson (1982);
Johannesson
et al. (1983);
Grahame et al.
(2006)
Johannesson
(1986)
Janson (1983);
Hull et al.
(1996); RolánAlvarez et al.
(1997);
Johannesson
et al. (2000);
Cruz & Garcia
(2001)
Johannesson et al.
(1995); Pickles &
Grahame
(1999);
Hollander et al.
(2005);
Johannesson
et al. (2008)
Rolán-Alvarez
et al. (1996);
Panova et al.
(2006);
Grahame et al.
(2006); Galindo
et al. (2009)
a
Personal observation.
Following the definition of Futuyama & Mayer (1980). All individuals are capable of crossing the hybrid zone.
Nm over hybrid zone compared with Nm over same distance within populations of pure ecotype.
b
c
Rockall ( Johannesson 1988). In addition, females of
L. saxatilis are strongly promiscuous and usually
carry sperm from eight to ten males (Mäkinen et al.
2007). This together with a rapid population growth
prevents a serious loss of genetic variation even if a
new site is colonized by only a single female (Nei
et al. 1975; Chakraborty & Nei 1977; Janson 1987a).
4. ECOTYPES OF LITTORINA SAXATILIS
A number of different ecotypes of L. saxatilis have been
described on the basis of morphological, ecological
Phil. Trans. R. Soc. B (2010)
and genetic relationships. The best investigated ones
are the S and E ecotypes (from Sweden), the H and
M (from the UK) and the RB and SU (from Spain),
however, additional ecotypes include the mudflat ecotype tenebrosa, the extremely small neglecta living in
empty barnacle shells and the subarctic groenlandica
(figure 1). Although earlier studies resulted in descriptions of more than 20 different species, based on the
genetic and morphological analyses all these are
currently considered ecotypes of L. saxatilis (for an
extensive review, see Reid 1996), however, adjacent
ecotypes are separated by relatively strong
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K. Johannesson et al.
Repeated evolution of isolation
Figure 1. Six ecotypes of Littorina saxatilis illustrating the extensive variation in shell size and form that is present within this
species. All the individuals are adults of representative size and shape. Upper row from left to right: ‘neglecta’—an ecotype living
in the empty shells of dead barnacles in the UK and France, ‘S’—a crab-resistant ecotype found on boulder shores along the
Swedish west coast, ‘E’—a wave-resistant ecotype confined to granite cliffs on the Swedish west coast. Lower row from left to
right: ‘groenlandica’—a large ecotype found in subarctic areas along with ecotypes of smaller size, ‘SU’—a wave-resistant Spanish ecotype and ‘RB’—a crab-resistant Spanish ecotype.
reproductive barriers (table 1). In principle, a distinct
ecotype of L. saxatilis is expected whenever the species
occupies a new microhabitat. This 1 : 1 relationship
between phenotype of L. saxatilis populations and
the microenvironment they inhabit suggests that phenotypic evolution is strongly linked to ecological
factors. Indeed, a microhabitat covering only a few
metres of shore embedded in another habitat (e.g. a
few boulders in a crevice surrounded by smooth
cliffs) will host a population of snails with a phenotype
matching the local boulder habitat.
In Sweden, Spain and the UK contrasting ecotypes
are present in what can simply be denoted as either
crab-rich or wave-swept intertidal habitats. Although
the particular habitats are found in somewhat different
intertidal contexts, the impact of crab crushing and
wave-surge select for either a snail with a large and
thick shell, a relatively small aperture and wary
behaviour, or a snail with a small and thin shell, a
relatively large aperture and bold behaviour (table 1).
An important piece of information is that differences
between ecotypes in morphology and behaviour are
largely inherited although a minor part of the difference is because of phenotypic plasticity (e.g. Janson
1982; Johannesson & Johannesson 1996; Carballo
et al. 2001; Hollander et al. 2006; Conde-Padı́n et al.
2007, 2009). In the UK and Spain, both ecotypes
are present on the same shores but at different tidal
heights. In the mid-shore, narrow zones of contact or
overlap, depending on the shore, are formed and
here hybrids are also present ( Johannesson et al.
1993; Hull et al. 1996). In Sweden, the ecotypes are
confined to different shores of contrasting microhabitats (boulder or cliffs) and in microhabitat shifts,
ecotypes overlap and hybrids are produced. The distribution of different microhabitats, and in particular, the
contrast of crab selection (selecting for large size, thick
shell and a snail that quickly retracts into the shell) and
Phil. Trans. R. Soc. B (2010)
wave-surf (selecting for small size to minimize drag
forces, large aperture to increase foot size and grip,
and a snail that rapidly emerges from the shell to
secure the grip to the substratum) provides the basis
of the existence of parallel phenotypes, in the first
place within each region (e.g. Sweden, Spain and the
UK) but, superficially at least, also among regions
(table 1).
5. EVIDENCES OF PARTIAL REPRODUCTIVE
ISOLATION
Estimates of gene flow outside and across hybrid zones
using genetic markers show that gene flow over hybrid
zones is only 10– 30%, or sometimes even less, of gene
flow over similar distances within continuous populations of the same ecotype (Rolán-Alvarez et al.
1996; Grahame et al. 2006; Panova et al. 2006;
Galindo et al. 2009). Both extrinsic and intrinsic
effects are likely to contribute to the impeded gene
flow. For example, immigrant inviability is likely to
be a major force preventing gene flow. That is, snails
crossing habitat borders (either by creeping or by rafting) reduce their chance of survival to about 10 –20%
of their earlier value ( Janson 1983; Rolán-Alvarez et al.
1997). In addition, parental ecotype snails disperse
non-randomly at least in the Spanish hybrid zone, contributing to the gene flow barrier (Erlandsson et al.
1999; Cruz et al. 2004). The presence of a postzygotic
component of isolation has been suggested from
observations from a hybrid zone in the UK, where
five hybrid females out of seven sampled had strongly
elevated levels of aborting embryos (Hull et al. 1996).
However, the abortion rate varies extensively among
females both within and among local populations of
any ecotype ( Janson 1985) and hence there are
alternative explanations for this observation that
cannot be rejected, such as simply an effect of a
Repeated evolution of isolation K. Johannesson et al.
small sample size, risk of cross-mating with a sibling
species (L. arcana), risk of sperm depletion owing to
very few mates being available (the UK hybrid zones
were reported to be of low density), or presence of
some local disease. In contrast, comprehensive data
of abortive embryos in samples from both Sweden
and Spain showed no indication of hybrid individuals
producing higher numbers of abnormal embryos
than other females ( Janson 1985; Johannesson et al.
2000). Hybrid male and female fecundity (another
indicator of possible intrinsic postzygotic isolation)
was similar to that of pure ecotypes in one study
(Johannesson et al. 2000), while slightly less in another
study (Cruz & Garcia 2001). Moreover, survival rates
of hybrids in the hybrid zone have been shown to be
higher, or as high as that of both parental ecotypes,
and higher than outside the zone, in both Spain and
Sweden (Janson 1983; Rolán-Alvarez et al. 1997),
while for the UK the evidence is less conclusive (table 1).
In both field and laboratory experiments, assortative mating of ecotypes is very strong at a local scale
as well as across regional scales, with barriers of
around 80 per cent isolation ( Johannesson et al.
1995a; Hull et al. 1996; Rolán-Alvarez et al. 1999;
Pickles & Grahame 1999; Cruz et al. 2004; Hollander
et al. 2005; Johannesson et al. 2008; Conde-Padı́n et al.
2008). In the laboratory, assortative mating is
observed at various stages of pair-formation and copulation. The first step is when males actively search for a
female to mate and use the mucous trails of other
snails to encounter a female (Erlandsson et al. 1999).
A male that encounters trails of females of two different ecotypes, more frequently follows trails of females
of his own ecotype ( Johannesson et al. 2008). The next
step of mating is when the male mounts the female and
inserts the penis under the female shell. Males of one
ecotype that have the possibility of mating with females
of two different ecotypes copulate with females of their
own ecotype for longer than the other females
(Hollander et al. 2005), and from studies of males
mating parasitized females, other males or juveniles
we know that short matings (less than 5 min) are
likely to represent failed copulation attempts with no
transfer of sperm, while a full-length mating is usually
around 20 –60 min (Saur 1990). Furthermore, laboratory experiments show that males mate longer with
females of the same ecotype from a population
150 km away than with females of a different ecotype
from an adjacent shore, despite the former being
genetically much more distantly related to the males
than the latter (Hollander et al. (2005), and see
Janson (1987b) for isolation-by-distance effects in
allozymes over geographical distances of 0 – 300 km).
Size is likely to be a major factor explaining the
assortative mating between ecotypes. In the field,
sizes of mates in a pair are positively correlated
(Johannesson et al. 1995a; Hull 1998; Conde-Padı́n
et al. 2008), indicating a general trend for sizeassortative mating. In fact, the observed degree of
assortative mating across seven geographically
distant populations was apparently predicted using
information on their size population variability
(Conde-Padı́n et al. 2008). As snails of crab-rich
environments (ecotypes RB, M and S, see table 1)
Phil. Trans. R. Soc. B (2010)
1739
are roughly twice the size of snails from wave-exposed
environments (SU, H and E, see table 1), size differences between ecotypes are likely to explain a major
part of their assortative mating. Indeed, if size differences are removed (by experiments in the laboratory
choosing extremely large and small individuals of ecotypes with different average sizes) mating barriers
largely disappear (Hollander et al. 2005; Johannesson
et al. 2008). Snails of different ecotypes have different
growth rates, and these are largely inherited, but also
extremely variable within populations ( Janson 1982;
Johannesson et al. 1997; Conde-Padı́n et al. 2007).
This suggests that there is a large amount of additive
genetic variation for selection to operate on. Indeed,
quantitative population differentiation estimates
(QST) for different shell shape and size traits between
sympatric ecotypes were significantly higher than molecular FST estimates (table 2 from Conde-Padı́n et al.
2007), suggesting that divergent natural selection is
mainly responsible for the inflated QST estimates
(Merilä & Crnokrak 2001). Most probably, crab predation favours rapid growth and large adult size
(Kemppainen et al. 2005), while wave surf selects for
slow growth and small adult size; a small size reduces
drag forces that can be excessive on rocky shores
(Denny 2000) and increases the availability of protective cracks (Raffaelli & Hughes 1978). Hence, size
could be a ‘magic trait’ (Gavrilets 2004) with pleiotropic effects on ecotype fitness and mate choice. Size
differences are under strong directional selection with
survival rates reduced to 10 – 20% of original values
when snails are transplanted between contrasting
environments (Janson 1983; Rolán-Alvarez et al.
1997), and as size is the major component of the phenotypic differences between ecotypes of the same area
(Sundberg 1988), selection is likely to act directly on
size. The exact mechanism of the size-based assortative mating remains unclear, but with respect to the
males’ tracking of females’ mucous trails, the width
of the trail will reflect the size of the female that laid
the trail, and males should be able to probe the trail
width. Both models of speciation (Gavrilets 2004)
and experimental trials (Rice & Hostert 1993) have
earlier shown that a magic trait facilitates the evolution
of a reproductive barrier in the face of gene flow, and
may be the most likely way new species are formed
in non-allopatric systems (Bolnick & Fitzpatrick
2007). Size is the dominant component of the difference between the two Swedish ecotypes (S and E,
Sundberg 1988) and is also likely to be the major
difference between ecotypes in the UK and Spain
(table 1).
To investigate the mechanisms of the repeated evolution of reproductive barriers, we need to unveil the
evolution of ecotype differences in size as well as in
any other traits contributing to the reproductive isolation. We might also be interested in the more
general issue of how similar ecotypes evolve repeatedly
in different sites, independent of the fact that they also
evolve reproductive barriers. A key issue for both these
questions is what mechanisms have been involved in
creating the ecotype differences we observe at present.
Have they all evolved in the presence of gene flow
between incipient ecotypes? Or, have both allopatric
Phil. Trans. R. Soc. B (2010)
haplotypes cluster by ecotype,
or, following extensive
introgression, by geographical
relatedness
ancestralf origin of ecotypespecific alleles. Double
colonization
in situ evolution of ecotypes
due to repeated
independent nonsynonymous substitutions
of the same nucleotide
in situ evolution of ecotypes
due to repeated
independent nonsynonymous substitutions
at different loci influencing
the same traits
(1) allopatry
(2A) non-allopatry
(parallel)—
parallel
evolution due to
same
substitutions
(2B) non-allopatry
(parallel)—
parallel
evolution due to
different
substitutions
as 2A
haplotypes cluster by
geographical relatedness (if
different lineages colonized
different areas), or haplotype
frequencies vary across
geographical areas due to
drift (as lineage sorting
progresses, the haplotype will
eventually cluster by
geographical area)
non-candidate loci
mechanism
model
b
as 2A
haplotypes cluster by
geographical relatedness (if
different lineages colonized
different areas), or haplotype
frequencies vary across
geographical areas due to
drift (as lineage sorting
progresses, the haplotype will
eventually cluster by
geographical area). Sites
closely linked to functional
substitutions affected by
independent local selective
sweeps
haplotypes cluster by ecotype.
Large variation within each
ecotype indicating old
lineages
candidate locic, neutral
variationd
unique non-synonymous
substitutions, correlated with
ecotype in each geographical
area. These substitutions
appear in different loci in
different geographical areas
same non-synonymous
substitutions, correlated with
ecotype, across geographical
areas. These substitutions
appear against different
genetic background (i.e.
sequence of a particular allele,
that acquired mutation) in
different geographical areas
same non-synonymous
substitutions, correlated with
ecotype, across geographical
areas
candidate loci, functional
variatione
differentiation among
geographically distant
populations greater at
candidate than at noncandidate loci
differentiation among
geographically distant
populations smaller at
candidate than at noncandidate loci
differentiation among
geographically distant
populations may be
smaller in candidate loci
because drift is opposed
by selection
candidate versus noncandidate loci
population differentiation
K. Johannesson et al.
phylogenetic patternsa
Table 2. Alternative mechanisms and their predicted genomic signatures for repeated evolution of ecotypes of Littorina saxatilis in a postglacial area (e.g. Sweden).
1740
Repeated evolution of isolation
Phil. Trans. R. Soc. B (2010)
alleles that are positively
selected in one ecotype are
present in low frequencies
as neutral or slightly
deleterious alleles in other
ecotypes including the
ancestral lineage that
invaded the area
recent mutations favoured by
selection in one habitat
spread relatively rapidly
and successively improve
local adaptation of the
different ecotypes
as 2A
as 2A
large haplotype variation in one
ecotype. Very low genetic
variation owing to recent
selective sweep of a derived
allele in the other ecotype
as 1
same non-synonymous
substitutions, correlated with
ecotype, across geographical
areas, at least in one ecotype.
These substitutions appear
against the same genetic
background (i.e. sequence of
a particular allele, that
acquired mutation) in
different geographical areas
for sites in close genomic
proximity, but different
backgrounds for unlinked
sites
as 1
b
Predominant patterns, stochastic variation among loci expected.
Nuclear genes, not affected by ecological selection and mtDNA.
c
Non-synonymous substitutions of coding regions (adaptive mutations of non-coding regions are not considered).
d
Introns, untranslated regions and synonymous substitions in coding sequences of candidate genes.
e
Non-synonymous substitutions in the coding sequence of candidate genes, possibly also mutations in regulatory regions, coupled with differential expression.
f
Pre-dating colonization.
a
(4) non-allopatry
(repeated)—
evolution in
concert
(3) non-allopatry
(repeated)
standing genetic
variation
differentiation among
geographically distant
populations much smaller
for candidate loci in one
ecotype (may vary among
loci) but not in the other
ecotype
as 1
Repeated evolution of isolation K. Johannesson et al.
1741
1742
K. Johannesson et al.
Repeated evolution of isolation
and parapatric or even sympatric elements been
involved in ecotype evolution? Existing genetic data
provide some input to this, as do the results of a
recent model (Sadedin et al. 2009), and we review
these below. However, a major step forward will
require linking phenotypic differentiation to patterns
of evolution at the molecular level (Colosimo et al.
2005; Rogers & Bernatchez 2005; Seehausen et al.
2008), and so we here propose specific hypotheses
that may explain repeated ecotype differentiation in
L. saxatilis, and from each of these we suggest testable
predictions that will be useful to design the next
generation of studies.
6. MECHANISMS OF ECOTYPE FORMATION
Using the two Swedish ecotypes (E and S) as an
example, here we propose different mechanisms for
their evolution, illustrating the various possible
alternative mechanisms that need to be considered.
The E and S ecotypes are confined to the Swedish
west coast and adjacent areas of southern Norway,
and they are present on wave-exposed boulder (S)
and cliff (E) shores of islands and mainland shores
(figure 2). The coast is a postglacial environment
that became available for marine species 10 000 years
ago, and since then postglacial uplift of land has
continuously formed new islands. The establishment
of L. saxatilis populations in this area has taken place
in two steps; the first step being colonization of the
first available habitat in the area, while the second
step (still ongoing) is the colonization of new emerging
islands.
A key question is where and when did the genetic
variation originate that shapes the E and S ecotypes,
including the differences that maintain their reproductive barrier. Here, we discuss four mechanisms that
alone or in various combinations are likely to have contributed to the formation of the two ecotypes and the
reproductive barrier:
(a) Allopatry
The essential formation of the two ecotypes predates
the colonization of Sweden and they were introduced
by double colonization from elsewhere (with minor
later modifications). Upon colonizing Sweden they
established secondary contact resulting in local introgression. Although the original evolution of the
separate ecotypes may, or may not, have been in allopatry, the Swedish establishment was essentially an
allopatric process.
(b) Parallel evolution
A single Swedish colonization followed by nonallopatric separation in local islands where genetic
differences between E and S evolved repeatedly by
parallel selection pressures acting on mutations of
local origin (parallel evolution). We can think of
either identical substitutions occurring repeatedly
and independently in different populations, or
unique mutations occurring in each location, but
these having similar effects on the phenotype (Wood
et al. 2005; Arendt & Reznick 2007).
Phil. Trans. R. Soc. B (2010)
(c) Standing genetic variation
Non-allopatric separation as in (b) but selection acting
on ancestral alleles present in low frequencies in the
first founder group that invaded the Swedish archipelago, or introduced later by long-distance migrating
snails. Specific alleles would rapidly increase in frequency whenever populations expand into new
microhabitats where these are favoured (Colosimo
et al. 2005; Barrett & Schluter 2007). This variation
could originally have evolved either in allopatry or in
non-allopatry, but the formation of E and S is based
on pre-existing variation.
(d) Evolution in concert
Non-allopatric separation as in (b) and (c) but the
variation shaping E and S has a recent origin, essentially originating in the evolving Swedish populations
by the accumulation of positively selected mutations
in each habitat that spread rapidly by selective
sweeps, and successively promoted the formation of
more extreme ecotypes (Rieseberg & Burke 2001;
Morjan & Rieseberg 2004).
From each of these mechanisms one may outline the
patterns that will be predicted in neutral as well as
adaptive parts of the genome (table 2). A limitation
with earlier attempts based only on multi-loci comparisons of neutral markers (see below) is that
extensive secondary introgression can erode ancestral
differences formed during earlier periods of allopatry.
However, differences in neutral sequences linked to
adaptive loci will be maintained in the presence of
hybridization, as selection impedes introgression of
these sequences. We therefore expect variation in
these neutral sequences to distinguish between ancestral
origin and double invasion, on the one hand, and recent
and repeated origins of ecotypes, on the other (table 2—
1 versus 2 and 4). However, if ecotypes are formed from
standing genetic variation, linked neutral sequences will
diverge in much the same way as under a double invasion (Barrett & Schluter 2007). Although in this case,
there will still be different phylogenetic patterns in
non-candidate loci (species trees) separating the two
models of evolution, unless extensive introgression has
removed allopatric differences under model 1
(table 2—1 versus 3). One additional expectation is
that differences between ecotypes in variation linked to
adaptive loci would extend much further (genomically)
in the allopatric case than it would in the standing
variation case. This is because recombination erodes
the linkage disequilibrium more effectively within a
population than between populations. Recently, it has
been suggested that genomic hitchhiking around key
QTLs may be an important effect in the early divergence
of species (Smadja et al. 2008; Via & West 2008).
It is important that the various models described in
table 2 must be considered under specified scales of
time or space (or both). For example, the independent
substitution of the same nucleotide in separate subareas along the Swedish coast may be considered as
an example of parallel evolution (model 2) within a
larger area (Sweden), while at a scale of local islands,
among which the new mutations spread, this will be
Repeated evolution of isolation K. Johannesson et al.
1743
boulders
Saltö
S ecotype
0
200 m
environmental shifts
hybrids
cliffs
E ecotype
Figure 2. The distribution of Littorina saxatilis S and E ecotypes and hybrid forms along a rocky shore of a Swedish island. This
island is rather typical for the Swedish west coast in that its shore consists of a mosaic of boulders and cliffs, and the distribution of the two ecotypes strictly follows the microhabitat distribution with hybrid zones where the habitat shifts.
an example of evolution in concert (model 4). Furthermore, it is important to stress that gene and
population trees will not coincide for all loci in any
of the models.
7. ALLOPATRY VERSUS NON-ALLOPATRY—
EXISTING DATA
Given the limitations of multi-locus techniques discussed above, here we summarize the conclusions
from the existing data. A major question that has
been addressed in several earlier studies is whether
the formation of the reproductively isolated ecotypes
in L. saxatilis is largely a result of allopatric separation
followed by secondary overlap and hybridization, or if
most of the genetic variation that separates ecotypes
(neutral as well as adaptive) has a non-allopatric
origin (model 1 versus 2 – 4). A number of studies
have used genetic markers (allozymes, microsatellites,
AFLP) to assess the differentiation within- and
between-ecotypes within a geographical area (UK:
Wilding et al. 2001; Grahame et al. 2006, Spain:
Johannesson et al. 1993; Rolán-Alvarez et al. 2004,
Sweden: Johannesson & Tatarenkov 1997; Panova
et al. 2006; Mäkinen et al. 2008). These analyses
show notably similar patterns; multi-locus comparisons based on allozymes, microsatellites or AFLP
variation cluster samples by geographical relatedness
rather than by ecotypes (e.g. figure 3a,b). Furthermore, in Spain, the same mtDNA haplotypes are
shared between ecotypes within the same region,
while haplotypes of different regions are widely separated in the phylogeny (Quesada et al. 2007; figure 3c).
In addition, candidate AFLP loci showed significantly
larger geographic differentiation than non-candidate
loci, which is in agreement with our expectations
from parallel evolution of reproductive isolation
(table 2).
Phil. Trans. R. Soc. B (2010)
To fit these data into a general allopatric model,
where ecotypes evolve only when physically isolated
from each other is complicated, and requires the complete removal of all differences accumulated during
earlier allopatric phases. Indeed, this requires extensive
secondary introgression between local ecotypes in
combination with periods of demographic fluctuations. Notably, extensive secondary hybridization
between allopatrically isolated lineages of bivalve
species (Mytilus edulis/trossulus/galloprovincialis, and
two subspecies of Macoma balthica) has not removed
differences in allozymes, nuclear and mitochondrial
genes accumulated during the earlier allopatric
phases (Riginos & Cunningham 2005, 2007; Nikula
et al. 2008).
Existing data hence support a non-allopatric model
of separation, but the data are superficial and inconclusive for individual loci, some of which may be
extremely important in the reproductive isolation of
ecotypes. Therefore, there is a need for a more
comprehensive phylogenetic analysis based on both
selected and neutral variation of nuclear genes, not
least because a mixed model including both allopatric
and non-allopatric elements might be expected.
8. A SCENARIO OF ECOTYPE FORMATION
To illustrate a largely non-allopatric path of ecotype
formation, let us consider the formation of E and S
ecotypes in Sweden. The snails that colonized
Sweden probably rafted here from the UK or France
(more adjacent North Sea coasts are dominated by
sandy beaches and suitable habitats are sparse), and
in the light of the biology of the species and the estimated Nm ¼ 0.03 between islands at 0.1 – 1 km
distance (see above), an educated guess is that Nm
for long-distance colonization should be at least one
order of magnitude smaller. As described above,
1744
K. Johannesson et al.
Repeated evolution of isolation
(a)
(b)
(c)
S
E
E
E
E
E
H
M
S
M
S
S
S
H
H
S
1
2
M
E
Figure 3. Genetic clustering of contrasting ecotypes of Littorina saxatilis from Sweden, the UK and Spain based either (a,b) on
multi-locus estimates of genetic distances (microsatellites and AFLP) or (c) inferred from the phylogeny of mtDNA. Ecotypes
are denoted with letters according to the terminology used in the different countries where S, M and RB are crab-tolerant phenotypes and E, H and SU are wave-tolerant (see table 1 for details). Samples from the same geographical sites (sampled within
50– 100 m distance) are encircled. Open circles, RB; closed circles, SU.
colonization is likely to be initiated by a single mated
female and a new population will expand rapidly to
population sizes of 10 000 snails or more (as
shown by empirical observations, Johannesson &
Johannesson (1995), and by modelling, Sadedin
et al. (2009)). Following the initial colonization, the
species probably quite rapidly colonized additional
areas along the coast, a process that would possibly
have been completed over some 100 years ago, assuming a step-wise pattern and Nm 0.01 between
adjacent sites. Assuming that long-distance migration
is much less likely than migration between nearby
islands along the Swedish coast, it seems likely that
after the establishment of the first island population,
other islands were colonized by females from this
island, and so forth. Indeed, colonization of new
islands that continued to emerge out of the sea is
still ongoing. The progress of this second step is
likely to start with the appearance of an uplifted
island in the form of a wave-swept cliff (an E habitat).
A rafted E ecotype female from a nearby larger island
is the most likely founder of this little island, and in a
few generations, gives rise to a population of several
thousands of snails (such as has been observed following repopulation events of small islands after a toxic
algal bloom, Johannesson & Johannesson 1995).
After the top of the island has emerged a few metres
above mean water, the first boulder habitat becomes
available. The only way to explain the common
origin of all snails within an island in neutral
markers described earlier is that the boulder habitat
is colonized by members of the already established
E ecotype population. This population will be
restricted by either the mutation rate (parallel evolution), the available genetic variation already present
(standing genetic variation) or genes introduced to
the population by rafting snails from a boulder habitat
already established in the area (evolution in concert).
In all of these models, the neutral variation on the
island will derive from the first colonization event,
and even if S alleles are introduced by single migrating
Phil. Trans. R. Soc. B (2010)
individuals thereafter, their effect on the pool of neutral alleles will be minor (as these events are likely to
be few in number when compared with the number
of snails of the established population). Hence, all
three parapatric models can explain the observations
referred to above, that contrasting ecotypes of the
same island share a pool of neutral genes.
9. A ROLE FOR PARALLEL EVOLUTION?
An individual-based model parameterized with data
from the Swedish E– S ecotype system shows that,
within an island, one ecotype can readily form from
the other under parapatric conditions over less than
1000 generations, under the assumption that the
mutation rate is 1025 and 2– 16 independently
evolving loci affect ecotype formation (Sadedin et al.
2009). Few loci with large effects rather than many
loci with small effects, moderate selection pressures
and the presence of intermediate zones provides the
most favourable conditions for the rapid formation of
ecotypes. Overall, the model supports parallel evolution of ecotypes by independent local mutations,
whenever migration contributing genetic variation
from other sources is low (Nm , 0.001).
Nevertheless, for adaptive alleles, extremely low
levels of gene flow may occasionally introduce alleles
that are selectively favoured and become important
to the new population. Field densities and dispersal
distances suggest Ne 10 000 in most populations of
L. saxatilis and thus loci with positive mutation rates
of 1027 or higher may possibly evolve by parallel evolution if Nm , 0.001, which seems likely to be the case
over large geographical distances (among countries),
while less likely at a scale separating islands within
an archipelago (or close localities along a coastline).
At a local geographical scale, parallel evolution seems
less likely unless mutation rates are high. Notably,
the evolutionary history of individual alleles can
only be reconstructed from gene trees linked to
Repeated evolution of isolation K. Johannesson et al.
phylogeographic information and not from allele frequency information alone.
10. PROSPECTS FOR THE FUTURE
Notably, it is likely that the formation of E and S
includes elements of two or more of these mechanisms, that is, some genes (possibly of allopatric
origin) may be ancestral and present as standing genetic variation in the founder group, while others
evolved more recently by repeated substitutions of
the same nucleotide in independent sites, or through
the rapid spread of new and strongly favoured alleles.
Simply for this reason—that we have to consider the
phylogenetic history and the function of individual
genes or regions of genes, one at a time—we need to
approach this problem using ‘omics-techniques’.
Today, genomics, transcriptomics, proteomics and
even metabolomics offer new approaches in nonmodel organisms that did not exist a few years ago
(Ellegren & Sheldon 2008). Genome- and transcriptome-wide sequencing generate large amounts of
data that can be used to develop panels of neutral markers or search for selected loci. The latter can be
identified, among other approaches, from associations
between ecotype and single variable nucleotides
(SNPs), by looking for genome regions with decreased
diversity, probably produced by selective sweeps, or by
comparing silent and non-silent substitution ratios in
candidate genes. For a single candidate gene, it is
also possible to relate sequence variation between
alleles to differences in their functional properties
(Andersen et al. 2009). Using the microarray technique, we can compare expression levels of many
genes in contrasting habitats or ecotypes, to estimate
divergence at the transcriptomic level and at the
same time identify more loci involved in ecotype differences (Ranz & Machado 2006; Shiu & Borevitz 2008).
Finally, combining genomics with methodologies
for quantitative traits may be the most successful
approach (Stinchcombe & Hoekstra 2008).
Our suggestion that size is a crucial factor involved
in the reproductive isolation of local ecotypes both in
Sweden and Spain (and probably also in the UK)
needs to be linked to knowledge on the genetic background of size. If we can identify dominant size
genes and analyse their phylogenetic history on a background of the neutral phylogeny and phylogeography,
we may finally be able to answer the question of
how the reproductive isolation between ecotypes
of L. saxatilis evolved, and hence cast new light on
an intriguing example of repeated evolution of
reproductive barriers.
This work was supported by a Linnaeus grant from the
Swedish Research Councils (VR and Formas, to K.J. and
C.A.), a grant from the MEC (CGL2008-00135/BOS) (to
E.R.A.) and an EU Transfer of knowledge grant (to R.K.B.).
We are grateful to Juan Galindo, John Grahame, Johan
Hollander, Humberto Quesada and Erik Svensson, and not
least to one anonymous reviewer, for discussions and
suggestions, and to Hans Ellegren and Staffan Ulfstrand
for organizing the Speciation Conference at Kristineberg,
Sweden.
Phil. Trans. R. Soc. B (2010)
1745
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